U.S. patent application number 15/829437 was filed with the patent office on 2019-04-25 for photopolymer composition and application thereof.
The applicant listed for this patent is NATIONAL YANG-MING UNIVERSITY. Invention is credited to Yuan-Min LIN, Jiun-Ming SU.
Application Number | 20190119429 15/829437 |
Document ID | / |
Family ID | 66169745 |
Filed Date | 2019-04-25 |
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United States Patent
Application |
20190119429 |
Kind Code |
A1 |
LIN; Yuan-Min ; et
al. |
April 25, 2019 |
PHOTOPOLYMER COMPOSITION AND APPLICATION THEREOF
Abstract
The present disclosure provides a photopolymer composition and
the applications thereof. The photopolymer composition comprises: 5
weight percent to 15 weight percent of gelatin methacrylate
(GelMA), 0.1 weight percent to 5 weight percent of silanized
biologically active additive, 0.1 weight percent to 5 weight
percent of photoinitiator, and 75 weight percent to 95 weight
percent of a solvent. Compared to a conventional hydrogel, the
hydrogel prepared from the photopolymer composition of the present
disclosure has improved compressive strength, mechanical strength
and stability. Accordingly, the hydrogel is applicable to
biomedical research and tissue repair.
Inventors: |
LIN; Yuan-Min; (Taipei,
TW) ; SU; Jiun-Ming; (Taipei, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL YANG-MING UNIVERSITY |
Taipei |
|
TW |
|
|
Family ID: |
66169745 |
Appl. No.: |
15/829437 |
Filed: |
December 1, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/222 20130101;
A61L 27/54 20130101; C08F 289/00 20130101; C12N 2537/10 20130101;
A61L 27/46 20130101; B33Y 10/00 20141201; C08F 290/141 20130101;
B33Y 70/00 20141201; C08F 290/141 20130101; C08F 220/08 20130101;
C08L 89/06 20130101; A61L 27/50 20130101; A61L 27/52 20130101; C08K
9/06 20130101; C12N 2529/10 20130101; C12N 5/0062 20130101; C12N
2533/30 20130101; C12N 2533/54 20130101; A61L 27/58 20130101; A61L
27/46 20130101; C08L 89/06 20130101; C08K 9/06 20130101; C12N
2513/00 20130101 |
International
Class: |
C08F 289/00 20060101
C08F289/00; A61L 27/22 20060101 A61L027/22; A61L 27/52 20060101
A61L027/52; A61L 27/58 20060101 A61L027/58; A61L 27/54 20060101
A61L027/54; C12N 5/00 20060101 C12N005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 19, 2017 |
TW |
106135956 |
Claims
1. A photopolymer composition, comprising: 5 weight percent to 15
weight percent of gelatin methacrylate (GelMA); 0.1 weight percent
to 5 weight percent of silanized biologically active additive; 75.1
weight percent to 5 weight percent of photoinitiator; and 75 weight
percent to 95 weight percent of a solvent.
2. The photopolymer composition according to claim 1, wherein the
silanized biologically active additive comprises silanized
hydroxyapatite, silanized .beta.-tricalcium phosphate (.beta.-TCP)
or silanized bio-active glass.
3. The photopolymer composition according to claim 1, the
photoinitiator comprises 2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)
propionamide].
4. The photopolymer composition according to claim 1, wherein the
photoinitiator can be excited by light with wavelength in a range
from 400 nm to 800 nm so as to induce photopolymerization.
5. The photopolymer composition according to claim 1, wherein the
solvent comprises water, phosphate buffered saline (PBS),
conditioned media from cell line or cell culture media.
6. A three-dimensional cell culture media, comprising the
photopolymer composition according to claim 1.
7. A tissue repair composition, comprising the photopolymer
composition according to claim 1.
8. A method for preparing a three-dimensional cell culture media,
comprising steps of: mixing 5 weight percent to 15 weight percent
of gelatin methacrylate (GelMA), 0.1 weight percent to 5 weight
percent of silanized biologically active additive, 0.1 weight
percent to 5 weight percent of photoinitiator into 75 weight
percent to 95 weight percent of a solvent to form a mixture; adding
at least one cell into the mixture; providing light to the mixture
to induce photopolymerization of the mixture comprising the cell;
and obtaining a three-dimensional cell culture media.
9. The method according to claim 8, wherein after the step of
adding at least one cell into the mixture, photopolymerization is
induced by a 3D printing apparatus to cure the mixture comprising
the cell.
10. The method according to claim 9, wherein during the step of
photopolymerization, the 3D printing apparatus provides light above
the mixture and cures the mixture based on modeling information to
form the three-dimensional cell culture media by sheet
lamination.
11. The method according to claim 8, the photoinitiator comprises
2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide] (VA-086).
12. The method according to claim 11, wherein the photoinitiator
can be excited by light with wavelength in a range from 400 nm to
800 nm so as to induce photopolymerization.
13. The method according to claim 8, wherein the cell comprises a
stem cell, a cancer stem cell, a cell line, a somatic cell or a
primary cell.
14. The method according to claim 8, wherein the silanized
biologically active additive comprises silanized hydroxyapatite,
silanized .beta.-tricalcium phosphate (.beta.-TCP) or silanized
bioglass.
15. The method according to claim 8, wherein the solvent comprises
water, phosphate buffered saline (PBS), conditioned media from cell
line or cell culture media.
Description
CROSS-REFERENCE TO RELATED APPLICATION
BACKGROUND OF THE INVENTION
Technical Field of the Invention
[0001] The present disclosure relates to a photopolymer
composition, and particularly relates to a photopolymer composition
applicable to cell culture, tissue regeneration and tissue repair
after formation.
Background
[0002] Tissue engineering is a kind of regenerative medicine, which
refers to the combination of clinical medicine, biological science,
and materials engineering to produce functional cells or tissues in
vitro. The functional cells or tissues repair or replace the
tissues to restore the aging or damaged tissues or organs in the
body. For tissue engineering, cells, scaffolds and bio-signaling
molecules are three major factors to determine the effectiveness of
tissue regeneration.
[0003] The selection of bio-signaling molecules is determined by
the cells or tissues to be prepared. For example, in order to avoid
immune rejection, cells are mostly derived from the autologous
cells from the human body, which are stem cells with
differentiation ability, such as mesenchymal stem cells. The stem
cells are co-cultured with specific bio-signaling molecules to
induce the cells to begin signaling transduction, which drives the
stem cell to differentiate into cells in specific tissue. Until
now, the knowledge of culture and isolation of stem cell and the
knowledge of bio-signaling molecules have been well developed. For
scaffold, which provides the cell an environment to grow,
relatively speaking, since factors such as the biocompatibility,
cell adhesion, and cytotoxicity of the scaffold affects cell
growth, which further affects the results of tissue preparation and
tissue regeneration, the researchers in this field are still
studying and improving the scaffold.
[0004] The selection of bio-signaling molecules is determined by
the cells or tissues to be prepared. For example, in order to avoid
immune rejection, cells are mostly derived from the autologous
cells from the human body, which are stem cells with
differentiation ability, such as mesenchymal stem cells. The stem
cells are co-cultured with specific bio-signaling molecules to
induce the cells to begin signaling transduction, which drives the
stem cell to differentiate into cells in specific tissue. Until
now, the knowledge of culture and isolation of stem cell and the
knowledge of bio-signaling molecules have been well developed. For
scaffold, which provides the cell an environment to grow,
relatively speaking, since factors such as the biocompatibility,
cell adhesion, and cytotoxicity of the scaffold affects cell
growth, which further affects the results of tissue preparation and
tissue regeneration, the researchers in this field are still
studying and improving the scaffold.
[0005] On the other hand, 3D printing has become more and more
popular. The modeling can be completed by drawing software or
three-dimensional scanner and then additively building up and
curing thin layers step by step under computer control. The
modeling can be finished without producing a real mold. 3D printing
is highly efficient in preparation and avoids the need for mold
preparation in advance, which may meet the demand of personalized
medicine. Therefore, 3D bioprinting is invented. The molding
methods of three-dimensional printing technology include
photo-curing, laser sintering, melt extrusion, etc. The material is
selected by the molding method. Among them, photo-curing method has
high accuracy and has good surface properties of finished products,
is applicable to a wide range of materials. Therefore, the
industries which are more demanding of the finished products prefer
to use photo-curing method. Photopolymer is used in photo-curing
method. During the method, light is provided as excitation energy.
A photoinitiator releases free radicals or cations after absorbing
light, which further drives the monomers to polymerize into a
polymer.
[0006] Accordingly, the purpose of the present disclosure is to
combine the characteristics of the Gelatin methacrylate (GelMA) and
the photopolymer, and further improve the structural strength and
the biofunctionality of the materials based on the biocompatibility
of the materials and the convenience of material formation. As a
result, the present disclosure provides a photopolymer composition.
When the photopolymer composition is applicable to the field of
tissue engineering, compared to a conventional photopolymer, the
photopolymer composition of the present disclosure has properties
such as devoid of cytotoxicity, with better compressive strength
and of providing bioactivity, thereby increasing its application to
biomedical research and tissue repair.
SUMMARY OF INVENTION
[0007] In one aspect, the present disclosure provides a
photopolymer composition comprising: 5 weight percent to 15 weight
percent of gelatin methacrylate (GelMA), 0.1 weight percent to 5
weight percent of silanized biologically active additive, 0.1
weight percent to 5 weight percent of photoinitiator, and 75 weight
percent to 95 weight percent of a solvent.
[0008] In one embodiment of the present disclosure, the silanized
biologically active additive comprises silanized hydroxyapatite,
silanized .beta.-tricalcium phosphate (.beta.-TCP) or silanized
bio-active glass.
[0009] In one embodiment of the present disclosure, the
photoinitiator comprises
2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide] (VA-086).
[0010] In one embodiment of the present disclosure, the
photoinitiator can be excited by light with wavelength in a range
from 400 nm to 800 nm so as to induce photopolymerization.
[0011] In one embodiment of the present disclosure, the solvent
comprises water, phosphate buffered saline (PBS), conditioned media
from cell line or cell culture media.
[0012] In another aspect, the photopolymer composition can be
further used for preparing three-dimensional cell culture
media.
[0013] In another aspect, the photopolymer composition can be
further used for preparing tissue repair composition.
[0014] Furthermore, the present disclosure provides a method for
preparing three-dimensional cell culture media. The method
comprises steps of: mixing 5 weight percent to 15 weight percent of
gelatin methacrylate (GelMA), 0.1 weight percent to 5 weight
percent of silanized biologically active additive, 0.1 weight
percent to 5 weight percent of photoinitiator into 75 weight
percent to 95 weight percent of a solvent to form a mixture; adding
at least one cell into the mixture; providing light to the mixture
to induce photopolymerization of the mixture comprising the cell;
and obtaining a three-dimensional cell culture media.
[0015] In one embodiment of the present disclosure, after the step
of adding at least one cell into the mixture, photopolymerization
is induced by a 3D printing apparatus to cure the mixture
comprising the cell.
[0016] In one embodiment of the present disclosure, during the step
of photopolymerization, the 3D printing apparatus provides light
above the mixture and cures the mixture based on modeling
information to form the three-dimensional cell culture media by
sheet lamination.
[0017] In one embodiment of the present disclosure, the
photoinitiator comprises 2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)
propionamide] (VA-086).
[0018] In one embodiment of the present disclosure, the
photoinitiator can be excited by light with wavelength in a range
from 400 nm to 800 nm so as to induce photopolymerization.
[0019] In one embodiment of the present disclosure, the cell
comprises a stem cell, a cancer stem cell, cell line, a somatic
cell or a primary cell.
[0020] In one embodiment of the present disclosure, the silanized
biologically active additive comprises silanized hydroxyapatite,
silanized .beta.-tricalcium phosphate (.beta.-TCP) or silanized
bio-active glass.
[0021] In one embodiment of the present disclosure, the solvent
comprises water, phosphate buffered saline (PBS), conditioned media
from cell line or cell culture media.
[0022] The three-dimensional cell culture media is prepared by the
photopolymer composition through the method for preparing
three-dimensional cell culture media of the present disclosure.
During the process of the photopolymerization of the photopolymer
composition, in addition to the crosslinkage between the monomers
of gelatin methacrylate, covalent bonds are also formed between
methacrylate group and silane group. As a result, the obtained
three-dimensional cell culture media has improved compressive
strength, has biocompatibility and without cytotoxicity at the same
time. Furthermore, the three-dimensional cell culture media
comprises hydroxyapatite, which is a biologically active material.
Therefore, the three-dimensional cell culture media of the present
disclosure can be broadly applicable to biomedical field and also
applicable to tissue regeneration and tissue repair.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a diagram showing the silanization of
hydroxyapatite by 3-Methacryloxypropyltrimethoxysilane
(.gamma.-MPS);
[0024] FIG. 2 is a diagram showing the structure of the polymer
(GelMA/HAp) after the polymerization of the photopolymer
composition of the present disclosure;
[0025] FIG. 3 is a process flow for the preparation of the
three-dimensional cell culture media of the present disclosure;
[0026] FIG. 4A is a X-ray photoelectron spectroscopy (XPS) spectrum
of the hydroxyapatite and the silanized hydroxyapatite, wherein the
peak that the symbol * indicate is Si 2p (Si--O), and the binding
energy is about 104 eV;
[0027] FIG. 4B are scanning electron microscopy (SEM) images of the
hydroxyapatite and the silanized hydroxyapatite respectively,
wherein the image is 50000.times. original magnification, and the
scale is 100 nm;
[0028] FIG. 5A is an attenuated total reflection Fourier-transform
infrared spectroscopy (ATR-FTIR) spectrum of the hydroxyapatite and
the silanized hydroxyapatite, wherein peak 1 is Si--CH3, peak 2 is
C.dbd.O (1638 cm-1), and peak 3 is C.dbd.C (1706 cm-1);
[0029] FIG. 5B is a chart showing the stress strain curves of 15
weight percent of gelatin methacrylate (GelMA), polymer of 15
weight percent of gelatin methacrylate (GelMA)/3 weight percent of
hydroxyapatite and polymer of 15 weight percent of gelatin
methacrylate (GelMA)/3 weight percent of silanized hydroxyapatite
respectively;
[0030] FIG. 5C is a chart showing the elastic modulus of 15 weight
percent of gelatin methacrylate (GelMA), polymer of 15 weight
percent of gelatin methacrylate (GelMA)/3 weight percent of
hydroxyapatite and polymer of 15 weight percent of gelatin
methacrylate (GelMA)/3 weight percent of silanized hydroxyapatite
respectively;
[0031] FIG. 5D are SEM images showing 10 weight percent of gelatin
methacrylate (GelMA), polymer of 10 weight percent of gelatin
methacrylate (GelMA)/3 weight percent of hydroxyapatite and polymer
of 10 weight percent of gelatin methacrylate (GelMA)/3 weight
percent of silanized hydroxyapatite respectively, wherein the scale
is 100 .mu.m, *, p<0.05, and ***, p<0.001; and
[0032] FIG. 6 are optical microscope results showing the cell
growth rate test of the mesenchymal stem cell of human encapsulated
by the three-dimensional cell culture media of the present
disclosure, wherein the image on the left shows the result after 6
hours of culture, the image on the right shows the result after 7
days of culture, and the scale is 100 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The following embodiments are given by way of illustration
to help those skilled in the art fully understand the spirit of the
present application. Hence, it should be noted that the present
application is not limited to the embodiments herein and can be
realized by various forms. Further, the drawings are not precise
scale and components may be exaggerated in view of width, height,
length, etc. Herein, the similar or identical reference numerals
will denote the similar or identical components throughout the
drawings.
[0034] Hydrogel is a material having both biocompatibility and
biodegradability. However, since hydrogel is highly
water-retentive, it is not liable to have fixed shape. Besides,
hydrogel is very unstable at human body temperature, that is,
hydrogel is difficult to maintain a gel or semi-solid form. In
order to apply hydrogel to the field of tissue engineering,
appropriate chemical modifications or specific chemical substances
are needed to change the properties of hydrogel. Accordingly, the
present disclosure provides a novel photopolymer composition
comprising hydrogel as a main component. The photopolymer
composition can be used for preparing three-dimensional cell
culture media and is applicable to tissue repair. The present
disclosure also provides a method for making a three-dimensional
cell culture media by the photopolymer composition. Exemplary
embodiments of the present application will be described in detail
with reference to the accompanying drawings hereafter.
[0035] The photopolymer composition of the present disclosure
comprises 5 weight percent to 15 weight percent of gelatin
methacrylate (GelMA), 0.1 weight percent to 5 weight percent of
silanized biologically active additive, 0.1 weight percent to 5
weight percent of photoinitiator, and 75 weight percent to 95
weight percent of a solvent.
[0036] In one embodiment, methacrylate (MA) is covalently bonded to
gelatin form gelatin methacrylate (GelMA) having multiple
carbon-carbon double bonds (C.dbd.C double bond). Besides, the
silanized biologically active additive is an additive modified by
silane group. The modification of silane group is to form multiple
hydroxyl groups on the biologically active material. The
biologically active additive may comprise material capable of
activating or stimulating cells or tissues. In one embodiment, the
biologically active additive comprises hydroxyapatite
(HAp),.beta.-tricalcium phosphate (.beta.-TCP) or bioglass, and
preferably, the biologically active additive comprises
hydroxyapatite for undergoing silanization modification to form
silanized hydroxyapatite (Si-HAp). Gelatin methacrylate can be
prepared by any chemical reactions known in the art and the details
will not be described herein. FIG. 1 is a diagram showing the
silanization of hydroxyapatite. In one embodiment,
3-Methacryloxypropyltrimethoxysilane (.gamma.-MPS) is used as a
coupling agent to induce hydroxyapatite to undergo silanization to
form silanized hydroxyapatite (Si-HAp).
[0037] As mentioned above, by using the photoinitiator, the gelatin
methacrylate and the silanized hydroxyapatite are bonded to form
polymer hydrogel. The photoinitiator releases free radicals or
cations after absorbing light, which further drives the monomers to
polymerize into a polymer. The selection of photoinitiator can be
determined by the wavelength range of light and the applications of
the polymer hydrogel afterwards. The wavelength range comprises
infrared light in a range from 760 nm to 1000 nm, visible light in
a range from 400 nm to 800 nm, and ultraviolet light in a range
from 10 nm to 400 nm. The applications can be grouped in to
biological need or non-biological need. In one embodiment, the
photoinitiator comprises azo-initiator, which can be excited by
visible light. In one embodiment, photoinitiator which can be
excited by light in a range between 350 nm and 480 nm or
photoinitiator which can be excited by blue light can be used.
Furthermore, 2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide]
(VA-086) is used, wherein
2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide] releases free
radicals or cations to induce the gelatin methacrylate and the
silanized hydroxyapatite to undergo photopolymerization after
excited by blue light with a wavelength of 440 nm.
[0038] Referring to FIGS. 1 and 2, in one embodiment of the present
disclosure, during the process of photopolymerization of the
gelatin methacrylate and silanized hydroxyapatite, in addition to
the cross linkage between the monomers of gelatin methacrylate,
carbon-carbon double bonds (C.dbd.C double bond) in the gelatin
methacrylate are also bonded to the hydroxyl group in the silanized
hydroxyapatite to form gelatin methacrylate/silanized
hydroxyapatite hydrogel (GelMA/Si-HAp hydrogel). As shown in FIG.
2, compared to the chemical-bonding network of the gelatin
methacrylate/hydroxyapatite hydrogel (GelMA/HAp hydrogel), the
chemical-bonding network of gelatin methacrylate/silanized
hydroxyapatite hydrogel (GelMA/Si-HAp hydrogel) is tighter.
Accordingly, compared to a conventional gelatin
methacrylate/hydroxyapatite hydrogel (GelMA/HAp hydrogel), the
gelatin methacrylate/silanized hydroxyapatite hydrogel(GelMA/Si-HAp
hydrogel) of the present disclosure has improved compressive
strength and stability.
[0039] Referring to FIG. 4, three-dimensional cell culture media
can be further prepared by the photopolymer composition of the
present disclosure. The method for making the three-dimensional
cell culture media comprises the steps of:
[0040] S10: mixing 5 weight percent to 15 weight percent of gelatin
methacrylate (GelMA), 0.1 weight percent to 5 weight percent of
silanized biologically active additive, 0.1 weight percent to 5
weight percent of photoinitiator, and 75 weight percent to 95
weight percent of a solvent to form a mixture;
[0041] S12: adding at least one cell into the mixture;
[0042] S14: providing light to the mixture to induce
photopolymerization of the mixture comprising the cell;
[0043] S16: obtaining a three-dimensional cell culture media.
[0044] In the step S10, 5 weight percent to 15 weight percent of
gelatin methacrylate (GelMA), 0.1 weight percent to 5 weight
percent of silanized biologically active additive, 0.1 weight
percent to 5 weight percent of photoinitiator are uniformly
dissolved in 75 weight percent to 95 weight percent of the solvent.
Specifically, the photopolymer composition can be put into water,
phosphate buffered saline (PBS), conditioned media from cell line
or cell culture media in advance and then an ultrasonic oscillator
is used to uniformly dissolve the aggregation to obtain the
mixture.
[0045] As mentioned in step S12, before the step of
photopolymerization of the mixture, the method further comprises
adding the cell into the mixture, uniformly mixing the cell and the
mixture, and adding a suitable cell culture media for the selected
cell to supply the cell with the material needed in cellular
growth.
[0046] As mentioned in step S14, the method comprises the step of
providing light to the mixture to excite the photoinitiator.
Specifically, the light is visible light in a range from 400 nm to
800 nm. In one embodiment, blue light in a range from 350 nm to 480
nm is used to excite the photoinitiator. In a preferably
embodiment, 2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide]
(VA-086) is used as the photoinitiator.
2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide] releases free
radicals or cations after excited by blue light with a wavelength
of 440 nm. Furthermore, after releasing free radicals or cations,
the gelatin methacrylate and the silanized hydroxyapatite are
induced to undergo photopolymerization and cured with the cells
together, which is called cell encapsulation. That is, the cells
are fixed in the gelatin methacrylate/silanized hydroxyapatite
hydrogel formed by photopolymerization of the mixture. As a result,
a three-dimensional environment more similar to the animal body is
provided for the cell to grow.
[0047] The cells comprise, but are not limited to, stem cells,
cancer stem cells, cell lines, somatic cells or primary cells. Any
cells that can live in vitro can be encapsulated in gelatin
methacrylate/silanized hydroxyapatite hydrogel to carry out cell
culture. On the other side, compared to the conventional method
comprising a step of using ultraviolet light to induce the
photopolymerization, since blue light is used to excite the
photoinitiator to induce the photopolymerization in the present
embodiment, damage to cells caused by the ultraviolet light during
the photopolymerization can be avoided. Accordingly, the
three-dimensional cell culture media prepared by the present
disclosure is more suitable to the biomedical field or tissue
regeneration for meeting the requirement of devoid of
cytotoxic.
[0048] If the cells encapsulated in the gelatin
methacrylate/silanized hydroxyapatite hydrogel are cell lines or
cancer stem cells, a three-dimensional environment in the animal
body can simulated in vitro for further study. If the cells
encapsulated in the gelatin methacrylate/silanized hydroxyapatite
hydrogel are stem cells with differentiation ability, the mixture
comprising the cells can be directly applied to any animal tissues,
and then light with specific wavelength is provided to irradiate
the mixture to induce the photopolymerization to fix the mixture on
the tissue. Accordingly, stem cells can differentiate into the
cells the same as that of the tissue under the induction of the
biologically active additive so as to undergo tissue repair.
[0049] Besides, different kinds of biologically active additives
can be added into the mixture comprising the photopolymer
composition. After the addition, the mixture can be directly
applied to any animal tissues, and then three-dimensional cell
culture media can be directly formed on the tissue after providing
light to irradiate the mixture to undergo tissue repair. In one
embodiment, silanized hydroxyapatite is added along with the stem
cells to prepare the gelatin methacrylate/silanized hydroxyapatite
hydrogel. The gelatin methacrylate/silanized hydroxyapatite
hydrogel is applied to the area of damaged bone, and then the
silanized hydroxyapatite, which has biologically active, induces
stem cells to differentiate into the bone cells to undergo tissue
repair.
[0050] In one embodiment of the present disclosure, before the step
S14 of photopolymerization of the mixture comprising the
photopolymer composition, the mixture is placed on a carrier of a
3D printing apparatus, and the light is adjusted to be over the
mixture. In this step, a desired model can be completed by drawing
software to obtain modeling information. After the modeling
information is input into the 3D printing apparatus, the 3D
printing apparatus can control the light in step S14 to move along
x, y, or z axis based on the modeling information to provide light
within a determined area so as to excite the photoinitiator in the
photopolymer composition. Accordingly, the mixture comprising the
photopolymer composition is gradually cured layer by layer to form
the desired model to obtain the three-dimensional cell culture
media.
[0051] Among the embodiments mentioned above, in order to meet the
requirements of the three-dimensional cell culture and the
application to the tissue repair, the mixture comprises 75 weight
percent of aqueous solution or cell culture media. Accordingly, the
mixture has good biocompatibility and is highly water-retentive.
Relatively speaking, the mixture has higher fluidity before
polymerization. As a result, light is configured to place over the
mixture so as to carry out photopolymerization within a determined
area, which removes the limitations of shaping the material when
the light is configured under the material in the conventional
way.
[0052] The three-dimensional cell culture media can be used to
culture stem cells, cancer stem cells, cell line, somatic cells or
primary cells in vitro so as to simulate three-dimensional
environment in a living body for further cell experiments in
biomedical research, such us cell growth test. Or, somatic cells or
primary cells can be culture to obtain the substance secreted by
the somatic cells or primary cells for further experiments or
applications to the living bodies. On the other side, the
three-dimensional cell culture encapsulating the stem cells can be
directly applied to the animal tissue. For example, the
three-dimensional cell culture media is directly applied to the
area of damaged bone. As a result, under the stimulation of
silanized hydroxyapatite, the stem cell is induced to differentiate
into bone cells and facilitates cell growth, which is helpful for
bone repair.
[0053] The following embodiments are given by way of illustration
to help those skilled in the art fully understand the spirit of the
present application. Hence, it should be noted that the present
application is not limited to the embodiments herein and can be
realized by various forms. Further, the drawings are not precise
scale and components may be exaggerated in view of width, height,
length, etc. Herein, the similar or identical reference numerals
will denote the similar or identical components throughout the
drawings.
[0054] The hydrogel of the present disclosure is provided by
polymerization of gelatin methacrylate and silanized
hydroxyapatite. The hydrogel of the present disclosure has improved
mechanical strength. The biocompatibility of the hydrogel is
further confirmed. Therefore, a photopolymer composition is
provided to undergo photopolymerization by the irradiation of
visible light. The photopolymer composition of the present
disclosure can be applicable to tissue repair and biomedical
research.
First Embodiment
Preparation of Gelatin Methacrylate Hydrogels
[0055] Gelatin methacrylate hydrogel is synthesized from gelatin
(type A, 300 Bloom). The method for making the gelatin methacrylate
hydrogels is described hereinafter. 5 grams of gelatin is added
into 50 mL of phosphate buffered saline (PBS). The gelatin and the
PBS are heated to a temperature of about 60.degree. C. The gelatin
is then dissolved completely in PBS. The gelatin solution is kept
under stirring condition. A total volume of 5 mL of methacrylate
anhydride (Sigma-Aldrich) is added into the gelatin solution at a
rate of 0.1 mL/min. The reaction is conducted for 3 hours at a
temperature of about 50.degree. C. 250 mL of phosphate buffered
saline at a temperature of about 50.degree. C. is added to
terminate the reaction. Modified polyethersulfone (PES) hollow
fiber membrane (diameter kDa MWCO, Spectrum Labs, USA) is used to
dialyze in distilled water at a temperature of about 50.degree. C.
for 24 hours to remove methacrylic acid. The obtained solution
after dialysis is then dried for 3 days to form a spongiform-like
product. The spongiform-like product is frozen at a temperature of
about -80.degree. C. Nuclear Magnetic Resonance (1H-NMR; Bruker,
Ascend 400, USA) is used to analyze the ratio of
methacrylation.
Second Embodiment
The Reaction of Silanization of Hydroxyapatite
[0056] 5 grams of hydroxyapatite nano powder (Sigma-Aldric), 0.1
gram of n-propylamine and 0.5 gram of
3-Methacryloxypropyltrimethoxysilane (.gamma.-MPS, Alfa Aesar) are
added into 100 mL of cyclohexane (Sigma-Aldrich). The resulting
solution is then agitated for approximately 30 minutes at a
temperature of about 25.degree. C. The resulting solution is then
heated to a temperature of about 60.degree. C. and kept for 30
minutes. Next, the resulting solution is placed into rotary
evaporator under a temperature of about .degree. 60 C. to remove
the solvent and then is heated at a temperature of about 90.degree.
C. for an hour. Finally, the powder is dried after being kept in a
vacuum oven for 72 hours.
[0057] After obtaining the silanized hydroxyapatite, X-ray
photoelectron spectroscopy (XPS, ESCALAB 250, Thermo Scientific,
USA) is used to analyze the composition. Referring to FIG. 4A,
compared to the pre-modified hydroxyapatite on the left, the peak
of Si with 2p orbital (Si 2p), which is indicated by the symbol *,
can be seen. The binding energy of Si is about 104 eV. Besides,
referring to FIG. 4B, scanning electron microscopy (JSM-7600F,
JEOL, USA) is used to observe the morphology of surface
modification. Silanization does not affect the morphology of
hydroxyapatite. Either hydroxyapatite granular or silanized
hydroxyapatite granular is still ball-like shape without
deformation.
[0058] Third Embodiment
Preparation of Mixture of Gelatin Methacrylate/Silanized
Hydroxyapatite (GelMA-Si-HAp)
[0059] Uniformly mixing 15 percent (w/v) of gelatin methacrylate, 1
to 3 percent (w/v) of silanized hydroxyapatite (Si-HAp) powder and
1 percent (w/v) of
2,2'-Azobis[2-Methyl-N-(2-hydroxyethyl)propionamide] (VA-086, Wako)
as a photoinitiator by an ultrasonic oscillator at a frequency of
20 kHz. Specifically, aggregation should be avoided in the
resulting mixture.
Fourth Embodiment
Mechanical Properties of Gelatin Methacrylate/Silanized
Hydroxyapatite Hydrogel (GelMA-Si-HAap Hydrogel)
[0060] After obtaining the mixture of gelatin
methacrylate/silanized hydroxyapatite from the third embodiment,
blue light with a wavelength of 440 nm (8 mW/cm.sup.2) is used to
irradiate the mixture for 1 minute to excite the photoinitiator,
which is VA-086 in the mixture, so as to induce
photopolymerization. Gelatin methacrylate/silanized hydroxyapatite
hydrogel without comprising any cells is obtained. The hydrogel is
then analyzed to realize the properties of chemical bonds and
mechanical properties.
[0061] Referring to FIG. 5A, Fourier-transform infrared
spectroscopy (ATR-FTIR) is used to analyze hydroxyapatite and
silanized hydroxyapatite (Si-HAp). In the spectrum of silanized
hydroxyapatite (Si-HAp), peak number 1 is Si--CH3 (870 cm-1), peak
number 2 is C.dbd.O (1706 cm-1), and peak 3 is C.dbd.C (1638 cm-1).
Accordingly, covalent bonds are proved to be formed between the
methacrylate group and the silane group, besides, with regard to
the mechanical properties, as shown in FIG. 5B, which shows the
stress strain curves. Compared to the hydrogel (GelMA-HAp)
polymerizing with 3% hydroxyapatite (HAp), the hydrogel
(GelMA-Si-HAp) polymerizing with 3% silanized hydroxyapatite
(Si-HAp) has better compressive Strength. Referring to FIG. 5C,
gelatin methacrylate/silanized hydroxyapatite hydrogel has better
elastic modulus. Further referring to FIG. 5D, the network of the
gelatin methacrylate/silanized hydroxyapatite hydrogel is tighter.
From the results, the properties and the morphologies of the
hydrogel mentioned above, the gelatin methacrylate/silanized
hydroxyapatite hydrogel (GelMA-Si-HAp) obtained from polymerization
of gelatin modified by methacrylate acid and the hydroxyapatite
modified by silane group, in addition to the crosslinkage between
the monomers of gelatin; covalent bonds are also formed between
methacrylate group and silane group. As a result, the network of
gelatin methacrylate/silanized hydroxyapatite hydrogel is tighter
and more solid, and thus has improved compressive strength.
Fifth Embodiment
[0062] Gelatin Methacrylate/Silanized Hydroxyapatite Hydrogel
(GelMA-HAp Hydrogels) is Prepared by 3D Printing Apparatus and
Cells are Encapsulated in the Hydrogel
[0063] Digital light processing projectors (D912HD, Vivitek,
Taiwan) is vertically set up above the sample tank and at a
distance of 10 cm. 1-3 mL of a mixture of gelatin
methacrylate/silanized hydroxyapatite (GelMA-HAp) comprising 1
weight percent of VA-086 as a photoinitiator is provided. A cell
number of 1.times.10.sup.6/mL of MG63 cell line or a cell number of
1.times.10.sup.7/mL of MSCs are mixed with the mixture. The
resulting mixture is uniformly distributed in the culture tray (9.6
mm.sup.2) On the other side, light-shielding are is designed in
advance by Power Point (Microsoft, USA). The digital light
processing projectors irradiates the area determined to be exposed
from top to down for 40 seconds to induce photopolymerization
within determined area of the gelatin methacrylate/silanized
hydroxyapatite (GelMA-HAp) comprising the cells to polymerize into
hydrogel. After irradiation, phosphate buffered saline is used to
wash away the unreacted materials. Gelatin methacrylate/silanized
hydroxyapatite hydrogel encapsulating the cells is obtained.
Sixth Embodiment
The Cell Growth Rate Test of the Mesenchymal Stem Cells of Human
Encapsulated in the Gelatin Methacrylate/Silanized Hydroxyapatite
Hydrogel
[0064] After obtaining the methacrylate/silanized hydroxyapatite
hydrogel encapsulating the cells from the fifth embodiment, the
hydrogel is observed after 6 hours of culture and after 7 days of
culture to analyze the cell grow capability. As shown in FIG. 6,
most of the mesenchymal stem cells encapsulated in the hydrogel are
still alive and distributed in the hydrogel after 7 days of
culture. Accordingly, the gelatin methacrylate/silanized
hydroxyapatite hydrogel of the present disclosure is devoid of
cytotoxicity. Therefore, the gelatin methacrylate/silanized
hydroxyapatite hydrogel of the present disclosure can be applicable
to biomedical research or tissue repair.
[0065] From the results of the embodiments mentioned above, the
present disclosure provides a photopolymer composition. During the
polymerization of gelatin modified by methacrylate group and
hydroxyapatite modified by silane group, in addition to the
crosslinkage between the monomers of gelatin, covalent bonds are
also formed between methacrylate group and silane group. As a
result, the obtained gelatin methacrylate/hydroxyapatite hydrogel
(GelMA/HAp hydrogel) has tighter chemical-bonding network.
Accordingly, the hydrogel of the present disclosure has improved
compressive strength and mechanical strength. Furthermore, the
photopolymer composition of the present disclosure comprises
photoinitiator excited by visible light. When the photopolymer
composition is applied to the biomedicine research or tissue repair
by the method for making the three-dimensional cell culture media
of the present disclosure, compared to the conventional method, it
is more convenient and efficient. Furthermore, the damage to the
cells can be avoided. Accordingly, the photopolymer composition and
the applications thereof and the method for making
three-dimensional cell culture media from the photopolymer
composition have advantages such as with improved mechanical
strength and applicable to tissue repair by comprising biological
active material.
* * * * *